Method for the determination of acid-base status in biological fluids



Sept. 16, 1969 Filed Dec. 29. 1965 s. K. PETERSEN ETAL 3,467,582 METHOD FOR- THE DETERMINATION OF ACID-BASB STATUS IN BIOLOGICAL FLUIDS 5 Sheets-Sheet 1 INVENTORS Geri Kakholm -Peier.sen

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United States Patent 3,467,582 METHOD FOR THE DETERMINATION OF ACID- BASE STATUS IN BIOLOGICAL FLUIDS Gert Kokholm Petersen, 31 Skovbrynet, Bagsvaerd, Denmark, and Erik B. Rasmussen, 7 Skovlodden, Holte, Denmark Filed Dec. 29, 1965, Ser. No. 517,370 Int. Cl. B01k 3/00 U.S. Cl. 204-1 12 Claims ABSTRACT OF THE DISCLOSURE A method for determining the acid-base condition of blood and the like wherein a liquid sample of the blood is introduced into a pH-Pco electrode for measuring these values. The Pco of the sample is then changed by exposing the sample to a gas having a substantially different P00 value. The Pco value of the gas need not be known, and the exact exposure time is not critical. The changed pH and Pco values are then determined, and the respective values, when placed on an acid-base nomogram provide for determination of the desired condition.

This invention provides an improved method for the determination of the acid-base conditions in biological fluids, mainly in plasma and blood.

Acid-base status in plasma and blood is traditionally described by three basic parameters, i.e. plasma bicarbonate (HCOf), Pco or CO tension, and the resulting value of pH.

Their relationship is expressed by the equation known as the Henderson-Hasselbalch equation:

in which the resultant pH derives from the sum of a dissociation constant pK, and the logarithm of the ratio of the bicarbonate radical (HCO3) or plasma bicarbonate, and the dissolved CO gas in the form of carbonic acid z 3)- In plasma associated with whole blood, and at its normal temperature of 38 C., the value of pK is generally accepted as 6.10; and the dissolved CO (H CO can be expressed as 0.03 Pco the figure Pco being the partial pressure of CO gas involved (in mm. of mercury) and 0.03 being a solubility factor related to 38 C. temperature. Accordingly, the classical Henderson-Hasselbalch equation can be rewritten:

a 1100; e 0.03 Pc0 2 In this manner, the three important and measurable values of the acid-base balance are disclosed for use in diagnosis and therapy. Thus, it can be seen that the resultant pH derives from a logarithm of a ratio between a value of non-respiratory origin (HCO and one of respiratory origin (Pco It is recognized that any complete and clinically usetul statement of acid-base balance must include the two values of pH and P00 and in addition, either the value of plasma bicarbonate (HCO -)or other parameters generally recognized as deriving from the value of plasma bicarbonate, such as Standard Bicarbonate, Buffer Base, or Base Excess.

It might be said that the three values needed to disclose completely the acid-base status from the equation (2) are the respiratory element (Pco the non-respiratory element plasma bicarbonate (HCO3 and the resultant pH. The non-respiratory element can be disclosed by either the value of plasma bicarbonate (HCO -)or other factors similarly expressive, such as Standard Bicarbonate,

pH=6.10+lo 3,467,582 Patented Sept. 16, 1969 Actual Bicarbonate Actual Bicarbonate is the plasma bicarbonate concentration measured in meq./l. plasma at the actual Pco and at the actual temperature. Generally this is calculated by measuring the values of pH and P00 and inserting these in Equation 2.

Standard Bicarbonate Standard Bicarbonate is the plasma bicarbonate concentration measured in meq./l. plasma at a Pco of 40 mm. Hg and at 37 C. or 38 C.

Base Excess (BE) The Base Excess measured in meq./l. of plasma or blood is defined as the amount of base needed to titrate the blood to an end-point of pH7.40 at Pco :40 mm. Hg and at 37 C. or 38 C. A positive value of Base Excess indicates an accumulation of base, while a negative value indicates an accumulation of acid.

Base Excess can be derived, by acceptable empirical relationships, from the value of plasma bicarbonate if one of the other values of pH and Pco is known, and the hemoglobin concentration of the blood is also known.

It is apparent from the above, and generally recognized, that the determination of acid-base statusrequiring a statement of the non-respiratory element (Standard Bicarbonate, Plasma Bicarbonate, or Base Excess), the respiratory element P00 and the resultant pH-can be accomplished by the following general measurements:

(a) The simultaneous measurement of pH and that of total CO (or plasma C0 The latter can be defined as the sum of the bound (non-respiratory HCOf) and the dissolved (respiratory) CO which can be calculated from:

with the calculation of the non-respiratory element from (2) above or from accepted nomographs generally used in the biochemical and medical profession.

In (a) above, Total CO is measurable by the Van Slyke manometric method which measures the Total CO in the blood plasma both as dissolved CO and as plasma bicarbonate. Together with a measurement of pH, this method results in accuracy of determination but is not generally acceptable due to the requirement for larger blood samples and experienced and skillful operators, and the equipment is more bulky and awkward to use.

Generally, the most satisfactory method is as outlined in (b) above, wherein pH is measured by means of a pH sensitive electrodea glass electrode; and a reference electrode. The actual Pco of the blood sample can be determined by means of a so-called Pco electrode, which generally consists of a glass electrode and a reference electrode mounted in a jacket, which contains a solution, in which the pH value depends on the Pco value in the said solution. The sensitive membrane of the glass electrode is covered with a thin layer of a porous material, over which a thin membrane of an electrically insulating plastic foil (for instance Teflon) is mounted, which permits an exchange of carbon dioxide between the sample, which is outside the membrance, and the solution at the sensitive membrane of the glass electrode inside the membrane. By measurement of the change of the pH 3 in the solution, a direct measure of the Pco of the sample is obtained.

The actual Pco of the sample can also be determined indirectly by an equilibration technique. This technique is based on the knowledge that corresponding values of pH and Pco always describe a straight line in a diagram, in which the pH value is the abscissa and log Pco is the ordinate.

According to this equilibration method the actual pH value of the sample is first measured. Then the sample is brought to an equilibrium with a C gas mixture with a known content of C0 The corresponding pH value is measured. Finally, the sample is brought to an equilibrium with another CO gas mixture with known content of CO but different from the first one, and the corresponding pH value is measured. The two pairs of corresponding values of pH and Pco are plotted in a pH, log Pco coordinate system as two points, through which a straight line is drawn. From the straight line and the measured actual pH value, the actual PCOg value of the sample is determined (see FIGURE 1).

If the straight line mentioned above is not drawn in a simple pH, log Pco coordinate system, but in the socalled Siggaard-Andersen Curve Nomogram (FIGURE 2), which in addition to the said axes has a standard bicarbonate axis, a base excess curve, and a buffer base curve, the standard bicarbonate can be found as the point of intersection between the straight line and the standard bicarbonate axis. The base excess and the butter base are found at the intersection between the straight line and the base excess curve and the butfer base curve, respectively.

The instruments which can be used for some or all of the determinations measured above have already been described in the literature and are available in many different forms. The simplest form of instrumentation is one which measures pH and Pco By this means pH and Pco can be measured, but the values of Standard Bicarbonate and/ or Base Excess do not result. This form of instrumentation is, therefore, only used to determine the respiratory condition of the blood sample, but not for the determination of the metabolic or non-respiratory condition of the blood sampe, which latter is indicated by the Standard Bicarbonate or Base Excess values.

Two other systems employ the previously described equilibration technique. One such system is mentioned in US. Patent No. 3,127,254 and is based on very small sample volumes (usually capillary blood). First the actual pH value of the sample is measured. Then the sample is brought to an equilibrium with a C0 gas mixture with known CO content, after which pH is measured. Then the sample is brought into equilibrium with another CO gas mixture, after which pH is measured. The equilibration takes place in a special equilibration system, which is designed in such a way that the CO gas mixture, in a thermostatted and humidified condition, is blown over the sample, which at the same time is vibrated quickly so that the surface of the sample is renewed very fast, which makes it possible to bring the equilibration time for blood down to three minutes without measurable hemolysis of the blood.

Another system is mentioned in US. Patent No. 3,147,- 081. According to this system, the pH value of the sample is first measured (sample volume approximately 0.5 1111.). Then a C0 gas mixture of known CO content is blown through the measuring chamber which contains the blood. The air stream will blow most of the blood out of the chamber, so that it forms a film which is brought to an equilibrium with the CO gas mixture, after which the blood collects again in the cuvette and the pH value is measured. Then another CO gas mixture of known CO Siggnard-Andersen, 0., & Engel, K.: A new acid-base nomogram. Scand. J. clin. Lab. Invest. 12, 177, 1960; and siggaardtndersen, 0. The pH-log PCOg blood acid-base nonwgram revised. Scand. J. clin. Lab. Invest. 14, 598, 1962.

4 content is blown through the chamber followed by another pH measurement when equilibrium is obtained. This system, in practice, is very diflicult to use, the equilibration of the blood film very often causing interruption in the measuring chain, and hemolysis of the blood often occurring.

The two equilibration systems mentioned above are the only systems by which the complete acid-base condition of the blood can be determined, but they both have definite disadvantages. It is necessary to make equilibra tions, and CO gas mixtures with precisely known CO content are necessary for this purpose.

An equilibration always requires a comparatively long time, because a substance is to be carried from one place to another. In this case, carbon dioxide is to be carried from the gas mixture to the liquid sample, or vice versa, until equilibrium is obtained. The speed of this transport is proportional to the difference in Pco in the gas mixture and in the liquid sample, which means that the speed decreases as the equilibrium is approached. Theoretically, it takes an indefinitely long time to obtain equilibrium, so there will always be a doubt as to accuracy when equilibrium is presumed to be complete. Blood, when warmed to 38 C., is rather unstable, as hemolysis easily takes place, and production of an acid by means of glycolysis can occur. It is very important to reduce the equilibration time.

Also, in such equilibration methods, gas supplies of precisely known CO content must be available, and the accuracy of the ultimate determination can only be as good as that of the CO content calibration of the gas cylinders. Gases of reliable calibration accuracy are often unavailable, or difiicult to obtain.

It is a general object of this invention to provide a new and improved method for the determination of acid-base status in biological fluids.

It is a more specific object of this invention to provide a method of the type described which is particularly suitable for the testing of blood samples.

It is a still further object of this invention to provide a method of the type described which is characterized by an extremely eflicient mode of operation, which can be carried out rapidly, and which does not depend upon the use of gases having precisely known compositions.

.Ihese and other objects of this invention will appear hereinafter, and for purposes of illustration, but not of limitation, the accompanying drawings illustrate aspects of the invention as follows:

FIGURE 1 comprises a plot illustrating values of pH and PCOg;

FIGURE 2 comprises a plot illustrating the coordinate system of FIGURE 1 modified with a Siggaard-Andersen curve Nomogram; and,

FIGURE 3 comprises a vertical sectional view of an electrode system which can be used in the practice of the instant invention.

As previously indicated, pH and P00; electrodes exist which can determine pH and Pco directly in a liquid sample. It has been found that the two electrodes can be mounted in a common chamber or close together so that a combined electrode system is created for the simultaneous measurement of pH and Pco As previously mentioned, one set of pH and P00 values is not suflicient to characterize the acid-base conditions in plasma or blood. At least one additional pair of corresponding values of pH and Pco is necessary to determine the straight line in the nomogram shown in FIGURE 2.

To obtain this additional set of corresponding values of pH and PCOg, the liquid sample is according to the invention influenced by a gas mixture during a suitable and comparatively short time with a Pco much diiferent from the Pco which is to be achieved in the sample. The action can be produced by simply bubbling the gas mixture through the liquid sample; or directing the stream of gas axially, radially, or tangentially towards the surface of the liquid with such a velocity that a fast renewal of the surface takes place. In addition to this, the stream of gas can be made to pulsate, which to a high degree improves the renewal of the surface. Furthermore, a fast renewal of the surface can be obtained by mechanical stirring of the liquid, for instance, by vibrating one of the electrodes. When a shift has been produced inside the sample and between sample and electrodes, the pH and Pco of the liquid sample are measured. Now available are two sets of pH-Pco values, one set of which is the actual values relating to the particular blood sample.

Most plasma and blood samples will have a PCOg close to 40 mm. Hg. To produce a change in the Pco of such a sample, a C0 gas mixture with a Pco of about 600 mm. Hg is suitable. It should be noted that the exact CO content of the gas mixture need not be known. In general, such a gas mixture will be suitable to change the Fee in the liquid sample from, for instance, 40 mm. Hg to about 60 mm. Hg. This change will take place about 100 times faster than in the case of a normal equilibration technique.

Instead of using a gas mixture with a very high Pco a gas mixture with a very low P00 can be used. Suitable gas mixtures, which can be used for high or low Pco are mixtures of oxygen and nitrogen, atmospheric air, carbon dioxide and oxygen, carbon dioxide and nitrogen, carbon dioxide-oxygen and nitrogen, carbon dioxide and atmospheric air, pure oxygen, pure carbon dioxide, and pure nitrogen. The change in Pco of a liquid sample from, for instance, 40 mm. Hg to 20 mm. Hg, by means of a gas mixture without CO will take place about seven times faster than according to the traditional equilibration method where equilibrium is defined in both cases as a condition where 99 percent of the maximum change in Pco has taken place.

Additional points on the line indicated in FIGURE 2 can easily be obtained according to the invention by repeating the influence by a gas mixture and subsequent measurement of pH and Pco The method according to this invention is thus characterized by the measurement of pH and Pco of a liquid sample, followed by a change in Pco and, therefore, also in the pH of the liquid sample by exposing the sample to a gas mixture, the Pco of which dififers from the Pco which is desired. Finally, corresponding values of pH and Pco are measured. By means of this method, the two previous disadvantages mentioned are eliminated. No time consuming equilibration has to be made, and no gas mixtures are used, in which the exact CO content must be known.

It is well known that pH and P00 electrodes require calibration. For the pH electrode a buffer of known pH, and for the Foo; electrode two CO gas mixtures with known Pco are used. While it is easy to make a buffer of known pH, the production of CO gas mixtures with known Pco values is difficult and time consuming. In addition, two such gases of known CO content, should they be used in the more traditional equilibration methods, would require lengthy equilibration times.

The method described in this invention also avoids other problems which characterizes certain known techniques. In the combined pH-Pco set-up which is used according to this invention, the pH electrode is calibrated by means of a buffer of known pH. Now a liquid sample with a known content of hydroxide, carbonate, or bicarbonate ions, is placed in the sample chamber. The liquid sample is subjected to a gas mixture with a high Pco by which hydroxide and carbonate ions are converted to bicarbonate ions. Equations which indicate the relation between pH and P00 for different solutions to be used can be calculated or determined from a number of measurements.

For instance, for a 20.00 mmol/l. NaOH solution at 38 C., the following relationship exists:

log Pco =l.006 pH+9.093

and for a saturated Ca(OH) solution at 38 C., the following relationship exists:

log Pco 1.58 pH+ 12.452

When the liquid sample has been exposed to the gas mixture, the pH value is measured and the corresponding Pco is derived from the equation, indicating the relation between pH and Pco Now the first standardization point for the Pco is available by means of the calibrated solu tion.

Now the liquid sample is exposed once more to the CO gas mixture; the pH value is measured; and the corresponding Pco is calculated. The second point of standardization for the Pco electrode is then available, by which the complete standardization procedure for the Pco electrode is completed.

The following provides an example describing the complete measuring procedure in accordance with the invention:

A buffer solution with pH:7.381 is placed in the combined pH-Pco measuring system working at 38 C., after which the pH electrode set-up is calibrated.

Then a 20.00 mmol/l. NaOH solution is placed in the measuring set-up, after which the solution is subjected to a C0 gas mixture with a Pco of about 600 mm. Hg.

Now the pH value is determined as being, for instance, 7.80according to which the first calibrated point of the Pco electrode should be set to Pco =17.7 mm. Hg. (The Pco is calculated according to the formula mentioned above.)

After that the solution is exposed to the CO gas once more; the pH value is determined again as 7.20, according to which the second calibration point of the Pco electrode is set to Pco =69.3 mm. Hg.

Now the electrode system is standardized.

The liquid sample, which is blood, in which the acidba se condition is to be determined, is placed in the measuring system.

The actual pH and Pco of the blood is measured directly (pH=7.35, Pco =35 mm. Hg). Now the blood is exposed to the CO gas mixture with a Pco of about 600 mm. Hg. Then pH and Pco are measured (pH=7.16, Pco =68 mm. Hg).

The two sets of corresponding values of pH and Pco are plotted in a nomogram as the one shown in FIG- URE 2.

Through the two points plotted, a straight line is drawn.

This line intersects with the standard bicarbonate axis in the point;

Standard bicarbonate=l9.6 meq./l.

and the base excess curve in the point:

BE:5.5 meq./l.

Furthermore the buffer base is determined to be 40.4 meq./l.

If it is desired, the magnitudes determined can be used for calculation of:

Meq./l. Actual bicarbonate 18.7 Total CO of plasma 19.8

Now the complete acid-base condition of the blood has been determined.

The method outlined by this invention can be used in connection with many already known designs of measuring systems. In the following an example is described of such a system, but it is pointed out that this is only an example. Any combined pH-Pco measuring equipment which can be used according to the method described is covered by this invention.

A suitable system for carrying out the method according to this invention is a pH-Pco electrode set-up as shown in FIGURE 3. The electrode set-up or parts thereof are described in detail in U.S. application Ser. No.

7 517,369 filed Dec. 29, 1965, and entitled Electrode System for Electro-Chemical Measurements in Solutions.

The electrode set-up consists of a Pco electrode 1 of conventional design placed as a cover in the measuring chamber 11. The glass electrode of the pH electrode chain 2 is mounted as the bottom in the measuring chamber, while the reference electrode 4 through the salt bridge 3 is connected to the measuring chamber through its side wall. To fix the glass electrode and the reference electrode in the desired positions in the electrode holder 6, they have been provided with a cap screw 7 and a cap nut 5, respectively. The wall of the measuring chamber is made up by a soft gasket 10 of a gas tight material, for instance butyl rubber. This gasket is provided with a hole, in which a small piece of tubing 12 has been placed, which establishes the connection between the salt bridge of the reference electrode and the measuring chamber. To as sure a constant temperature during the measurements, the complete electrode holder 6 is mounted in a jacket 9 through which thermostatted water is circulated, O-rings 8 sealing the ends of the electrode holder.

When the electrode set-up is to be used, the Pco electrode 1 is lifted up from the electrode holder 6. The Pco electrode is placed for instance in a thermostatted container. Now the liquid sample, the pH and Pco of which is to be determined, is placed in the measuring chamber 11, then the Pco electrode is returned to the electrode holder 6 again. Now the pH and Pco are measured.

To change the Pco of the liquid sample, the Pco electrode is lifted up from the electrode holder 6, after which the stop er 13 is placed in the electrode holder. The stopper 13 is provided with an inlet tube 14, through which the gas mixture for changing the Pco of the liquid sample is conducted. The gas mixture escapes through the hole in the stopper. After the sample has been exposed to the gas mixture, the stopper is removed and replaced by the P00 electrode, after which the pH and Pco of the liquid sample are measured again.

The gas mixture used to change the Pco of the liquid sample can by a simple modification of the inlet tube 14 be applied at the temperature of the thermostatted jacket, for instance by placing the inlet tube inside a tube in which thermostatted water is circulated. Furthermore, the gas mixture can be conducted through a humidifier, by which the gas mixture is saturated with water vapor at the temperature of the thermostatted jacket.

In some pH-Pco electrode set-ups, it is not practical to expose the sample to the gas inside the measuring chamber. Furthermore, in some cases, the samples on which the pH and Pco are measured may be contaminated e.g. by a potassium chloride solution in the salt bridge.

In the first case, the sample can be removed from the pH-Pco electrode set-up and exposed to the gas in a suitable container outside the set-up and next placed in the set-up again, whereafter the pH and Pco are measured. In the other case, the sample can be divided into at least two parts. The pH and Pco of one part is measured in the pH-Pco electrode set-up and next removed.

The Pco of the other part of the sample is changed by exposing it to a gas in a suitable container outside the set-up and next placed in the set-up again, whercafter the pH and Pco are measured.

It will be understood that various changes and modifications may be made in the method described which provide the characteristics of this invention without departing from the spirit thereof particularly as defined in the following claims.

That which is claimed is:

1. A method for the determination of the acid-base condition in biological fluids comprising the steps of measuring the pH and Pco of a liquid sample in a pH-Pco electrode set-up, changing the Pco of the liquid sample by exposing the sample to a gas having a Pco value which is substantially different than the Pco value of said liquid sample but which is not exactly known, continuing said exposure for a time sutficient to change the pH and Pco of said liquid sample but insufiicient to bring about equilibartion of said sample and said gas, subsequently measuring the changed pH and Pco of the liquid sample, and applying the measured values to an acid-base measuring means.

2. A method according to claim 1 wherein the liquid sample after initially being exposed to the gas mixture, and after the pH and Pco values are taken, is exposed at least one additional time to said gas, the duration of the second exposure being different than the duration of the first exposure to thereby secure different values of pH and Pco and wherein pH and Pco values are measured after each exposure.

3. A method according to claim 1 including the steps of adjusting the temperature of said gas to the temperature of said electrode set-up before exposure of said sample.

4. A method according to claim 1 including the step of saturating said gas with water vapor at the temperature of the electrode set-up prior to conducting of said gas to said set-up.

5. A method according to claim 1 wherein said sample is exposed to said gas by bubbling the gas through the liquid sample.

6. A method according to claim 1 wherein said sample is exposed to said gas by blowing the gas over the surface of the liquid sample.

7. A method according to claim 1 wherein said gas is conducted to the liquid sample by means of a pulsating gas stream.

8. A method according to claim 1 wherein said gas comprises a member from the group of mixtures consisting of oxygen and nitrogen, atmospheric air, carbon dioxide and oxygen, carbon dioxide and nitrogen, carbon dioxide-oxygen and nitrogen, and carbon dioxide an atmospheric air.

9. A method according to claim 1 wherein said gas comprises a mixture consisting of pure oxygen, pure carbon dioxide, or pure nitrogen.

10. A method according to claim 1 wherein the sample after having been initially measured is removed from the electrode set-up and exposed to the gas outside of the setup, thereafter replacing the sample in the set-up, and then measuring the changed pH and Pco of the sample.

11. A method according to claim 1 wherein the sample is divided into at least two parts, initially measuring the pH and Pco of one of said parts, removing said one part from the set-up, exposing the second part to said gas outside the set-up, placing said second part in said set-up, and measuring the changed pH and Pco of said second part.

12. A method according to claim 1 wherein a liquid is employed .for the calibration of the Pco electrode, said liquid being produced by placing an alkaline solution containing known concentrations of at least one member selected from the group consisting of hydroxide, carbonate, and bicarbonate ions in a pH-Pco, electrode set-up, and converting the hydroxide and carbonate ions present to bicarbonate ions by exposing the alkaline solutions to a gas containing CO References Cited UNITED STATES PATENTS 3/ 1964 Astrup et al 23-230 XR 9/ 1964 Stevenson et a1.

US. Cl. X.R. 

